Study of the effect of soy proteins on the acid-induced gelation of casein micelles

The objective of this research was to understand whether addition of soy protein to milk protein affects the properties of acid-induced casein gels. Different samples were prepared by suspending casein micelles pellets in milk serum containing soy proteins or whey proteins as well as mixtures of the two proteins. Glucono-delta-lactone was added, and the changes in apparent size (in diluted systems) as well as the viscoelastic properties of the mixtures were measured. Size exclusion chromatography was also carried out to characterize the soluble phase of the various mixtures before and after heating. Soy protein affected the gelation of the mixtures; however, not to the same extent as whey proteins, which dominated formation of the network in soy-whey-casein systems. It was concluded that, up to a critical ratio of soy/whey proteins, soy proteins can be incorporated in the mix without a significant change in structure of the casein gels.     

Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase

Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.     

Control of heat-induced aggregation of whey proteins using casein

The ability of alphas1/beta-casein and micellar casein to protect whey proteins from heat-induced aggregation/precipitation reactions and therefore control their functional behavior was examined. Complete suppression (>99%) of heat-induced aggregation of 0.5% (w/w) whey protein isolate (pH 6.0, 85 degrees C, 10 min) was achieved at a ratio of 1:0.1 (w/w) of whey protein isolate (WPI) to alphas1/beta-casein, giving an effective molar ratio of 1:0.15, at 50% whey protein denaturation. However, in the presence of 100 mM NaCl, heating of the WPI/alphas1/beta-casein dispersions to 85 degrees C for 10 min resulted in precipitation between pH 6 and 5.35. WPI heated with micellar casein in simulated milk ultrafiltrate was stable to precipitation at pH>5.4. Protein particle size and turbidity significantly (P相似文献   

Formation of soluble and micelle-bound protein aggregates in heated milk

The formation of heat-induced aggregates of kappa-casein and denatured whey proteins was investigated in milk-based dairy mixtures containing casein micelles and serum proteins in different ratios. Both soluble and micelle-bound aggregates were isolated from the mixtures heated at 95 degrees C for 10 min, using size exclusion chromatography. Quantitative analysis of the protein composition of the aggregates by reverse phase high-performance liquid chromatography strongly suggested that primary aggregates of beta-lactoglobulin and alpha-lactalbumin in a 3 to 1 ratio were involved as well as kappa-casein, and alpha(s2)-casein in micellar aggregates. The results gave evidence that heat-induced dissociation of micellar kappa-casein was implicated in the formation of the soluble aggregates and indicated that a significant amount of kappa-casein was left unreacted after heating. The average size of the aggregates was 3.5-5.5 million Da, depending on the available kappa-casein or the casein:whey protein ratio in the mixtures. The size and density of these aggregates relative to those of casein micelles were discussed.     

Interactions of whey proteins during heat treatment of oil-in-water emulsions formed with whey protein isolate and hydroxylated lecithin

The interactions of proteins during the heat treatment of whey-protein-isolate (WPI)-based oil-in-water emulsions with and without added hydroxylated lecithin were studied by examining the changes in droplet size distribution and the quantity and type of adsorbed and unadsorbed proteins. Heat treatment at 90 degrees C of WPI emulsions resulted in an increase in total adsorbed protein; unadsorbed beta-lactoglobulin (beta-lg) was the main protein interacting with the adsorbed proteins during the first 10 min of heating, but after this time, unadsorbed alpha-lactalbumin (alpha-la) also associated with the adsorbed protein. In emulsions containing hydroxylated lecithin, the increase in total adsorbed protein during heat treatment was much lower and the unadsorbed beta-lg did not appear to interact with the adsorbed proteins during heating. However, the behavior of alpha-la during heat treatment of these emulsions was similar to that observed in the emulsions containing no hydroxylated lecithin. In the presence of NaCl, the particle size of the emulsion droplets and the quantities of adsorbed protein increased markedly during heating. Emulsions containing hydroxylated lecithin were less sensitive to the addition of NaCl. These results suggest that the binding of hydroxylated lecithin to unfolded monomers or intermediate products of beta-lg reduces the extent of heat-induced aggregation of beta-lg and consequently decreases the interactions between unadsorbed beta-lg and adsorbed protein. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated whey protein and hydroxylated lecithin solutions.     

Uniaxial compression of thermal gels based on microfluidized blends of WPI and heat-denatured WPI.

The effect of heat-denatured whey protein isolate (dWPI)/whey protein isolate (WPI) ratio (0-0.6), microfluidization pressure (0-1000 bar), and number of passes (1-10) on the uniaxial shear stress at 10% (sigma(10)) and 80% (sigma(80)) relative deformation of dWPI/WPI heat-induced gels (14% total protein, w/w) was studied. No correlation between the average diameter of aggregates and the dWPI/WPI ratio, microfluidization pressure, or number of passes was found. However, increasing the microfluidization pressure or the number of passes resulted in a narrower size distribution of aggregates. Increasing the dWPI/WPI ratio and the number of passes resulted in a decrease and an increase of gel hardness, respectively. The results were interpreted in terms of more random aggregation/gelation of proteins in the presence of aggregates that could result in localized heterogeneities into gels and more dissipation of the deformation energy during compression. The positive effect of the number of passes on the gel hardness was also considered to be due to a more homogeneous aggregation/gelation of proteins in the presence of smaller aggregates.     

Physicochemical properties of soy protein: effects of subunit composition

Soy protein elastomer (SPE) exhibits elastic, extensible, and sticky properties in its native state and displays great potential as an alternative to wheat gluten. The objective of this study was to better understand the roles of soy protein subunits (polypeptides) contributing to the functional properties of SPE. Six soy protein samples with different subunit compositions were prepared by extracting the proteins at various pH values on the basis of the different solubilities of conglycinin (7S) and glycinin (11S) globulins. Soy protein containing a large amount of high molecular weight aggregates formed from α' and α subunits exhibited stronger viscoelastic solid behavior than other soy protein samples in terms of dynamic elastic and viscous modules. Electrophoresis results revealed that these aggregates are mainly stabilized through disulfide bonds, which also contributed to higher denaturation enthalpy as characterized by DSC and larger size protein aggregates observed by TEM. Besides, the most viscoelastic soy protein sample exhibited flat and smooth surfaces of the protein particles as observed by SEM, whereas other samples had rough and porous particle surfaces. It was proposed that the ability of α' and α to form aggregates and the resultant proper protein-protein interaction in soy proteins are the critical contributions to the continuous network of SPE.     

Protein-peptide interactions in mixtures of whey peptides and whey proteins

The effects of several conditions on the amounts and compositions of aggregates formed in mixtures of whey protein hydrolysate, made with Bacillus licheniformis protease, and whey protein isolate were investigated using response surface methodology. Next, the peptides present in the aggregates were separated from the intact protein and identified with liquid chromatography-mass spectrometry. Increasing both temperature and ionic strength increased the amounts of both intact protein and peptides in the aggregates. There was an optimal amount of added intact WPI that could aggregate with peptides, yielding a maximal amount of aggregated material in which the peptide/protein molar ratio was around 6. Under all conditions applied, the same peptides were observed in the protein-peptide aggregates formed. The dominant peptides were beta-lg AB [f1-45], beta-lg AB [f90-108], and alpha-la [f50-113]. It was hypothesized that peptides could form a kind of glue network that can include beta-lactoglobulin via hydrophobic interactions at the hydrophobic binding sites at the surface of the protein.     

Rheological properties and characterization of polymerized whey protein isolates.

Whey protein polymers were formed by heating whey protein isolate solutions at 80 degrees C. Flow behaviors of whey protein polymers produced from different protein concentrations and heating times were comparable to various flow behaviors of hydrocolloids. Polymer formation was found to be a two-phase process. The initial protein concentration was a significant factor that determines the size and/or shape of the primary polymer in the first phase as shown by intrinsic viscosity. Heating time was a factor in determining the aggregation in the second phase as shown by apparent viscosity. Intrinsic viscosity of whey protein polymers was as high as 141.7 +/- 7.30 mL/g, compared to 5.04 +/- 0.20 mL/g for native whey proteins. The intrinsic viscosity and gel electrophoresis data suggested that disulfide bonds played an important role in whey polymer formation.     

Fibril assemblies in aqueous whey protein mixtures

Fibril formation in mixtures of whey proteins upon heating at pH 2 was investigated. Fibrils were found to coexist with other structures, such as spherulites. These spherulites consist of radially oriented fibrils. At total protein concentrations above 6 wt %, transparent gels were formed. Changing the ratio between the various whey proteins did not affect this gelation concentration as long as beta-lactoglobulin (beta-lg) was present, suggesting that beta-lg was dominant in the gelation. Pure alpha-lactalbumin and pure bovine serum albumin did not form fibrils, nor did they gel upon heating at pH 2 and 80 degrees C for up to 10 h. They did however induce a decrease in the beta-lg concentration needed for gel formation upon heating at pH 2. Our results suggest that beta-lg is the only fibril forming protein at the conditions used and that no mixed fibrils are formed.     

Modification of the rheological properties of whey protein isolate through the use of an immobilized microbial transglutaminase

A process was developed in which calcium-independent, microbial transglutaminase (mTgase) was immobilized to controlled-pore glass. Avidin was adsorbed to glass beads that had been derivatized and biotinylated. The enzyme was biotinylated and adsorbed to the avidin affinity matrix. Solutions of 8% whey protein isolate (WPI) were then incubated with the mTgase beads, resulting in limited cross-linking of whey proteins. As incubation time increased, intrinsic viscosity increased, gelation temperature decreased, and stronger, more brittle gels were formed upon heating. This process allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of cross-linking. The functional properties of several batches of WPI were modified using <10 mg of the same enzyme, illustrating the capacity of immobilized enzymes to be used more frequently in applications of this nature.     

Formulation engineering can improve the interfacial and foaming properties of soy globulins

In this contribution, we have analyzed the effect of different strategies, such as change of pH (5 or 7) or ionic strength (at 0.05 and 0.5 M), and addition of sucrose (at 1 M) and Tween 20 (at 1 x 10(-4) M) on interfacial characteristics (adsorption, structure, dynamics of adsorption, and surface dilatational properties) and foam properties (foam capacity and stability) of soy globulins (7S and 11S at 0.1 wt %). We have observed that (1) the adsorption (presence of a lag period, diffusion, and penetration at the air-water interface) of soy globulins depends on the modification in the 11S/7S ratio and on the level of association/dissociation of these proteins by varying the pH and ionic strength (I), the effect of sucrose on the unfolding of the protein, and the competitive adsorption between protein and Tween 20 in the aqueous phase. The rate of adsorption increases at pH 7, at high ionic strength, and in the presence of sucrose. (2) The surface dilatational properties reflect the fact that soy globulin adsorbed films exhibit viscoelastic behavior but do not have the capacity to form a gel-like elastic film. The surface dilatational modulus increases at pH 7 and at high ionic strength but decreases with the addition of sucrose or Tween 20 into the aqueous phase. (3) The rate of adsorption and surface dilatational properties (surface dilatational modulus and phase angle) during adsorption at the air-water interface plays an important role in the formation of foams generated from aqueous solutions of soy globulins. However, the dynamic surface pressure and dilatational modulus are not enough to explain the stability of the foam.     

Effect of soy milk characteristics and cooking conditions on coagulant requirements for making filled tofu

The amount of coagulant added to soy milk is a critical factor for tofu-making; particularly it affects the textural properties of tofu. Earlier research indicated that the critical point of coagulant concentration (CPCC) is a characteristic parameter of soy milk and could be used as an effective indicator of optimal coagulant concentration (OCC) for making filled tofu. The objective of this study was to investigate the possible correlations between CPCC and the characteristics of soy milk made from various soybean samples and the effect of soy milk cooking and dilution conditions on CPCC. CPCC was determined by a titration method. Calcium chloride and magnesium chloride were used as coagulants. Soy milk characteristics including solid, protein, phytate, pH, titratable acidity, mineral content, and 11S/7S protein and these characteristics as affected by heating rate, heating time, and sequence of dilution and heating were studied. The results showed that the CPCC was significantly (p < 0.05) positively correlated with phytate content (grams per gram of protein), pH, and 7S protein content but negatively correlated with protein content, 11S protein content, 11S/7S ratio, titratable acidity, and original calcium content. Within the same soybean material, more proteins required more coagulant, but higher protein concentration during cooking resulted in less coagulant required by each gram of protein during coagulation. The CPCC decreased with increasing soy milk heating time or decreasing heating rate. The sequence of heating and diluting for preparing soy milk also had an effect on CPCC.     

Heat-induced changes in the ultrasonic properties of whey proteins

The physical aggregation of commercial whey protein isolate (WPI) and purified beta-lactoglobulin was studied by ultrasound spectroscopy. Protein samples were dialyzed to achieve constant ionic strength backgrounds of 0.01 and 0.1 NaCl, and gelation was induced in situ at constant temperatures (from 50 to 75 degrees C) or with a temperature ramp from 20 to 85 degrees C. Changes in the ultrasonic properties were shown in the early stages of heating, at temperatures below those reported for protein denaturation. During heating, the relative ultrasound velocity (defined as the difference between sample velocity and reference velocity) decreased continuously with temperature, indicating a rearrangement of the hydration layer of the protein and an increase in compressibility of the protein shell. At temperatures <50 degrees C the ultrasonic attenuation decreased, and <65 degrees C both velocity and attenuation differentials showed increasing values. A sharp decrease in the relative velocity and an increase in the attenuation at 70 degrees C were indications of "classical" protein denaturation and the formation of a gel network. Values of attenuation were significantly different between samples prepared with 0.01 and 0.1 M NaCl, although no difference was shown in the overall ultrasonic behavior. WPI and beta-lactoglobulin showed similar ultrasonic properties during heating, but some differences were noted in the values of attenuation of WPI solutions, which may relate to a less homogeneous distribution of aggregates caused by the presence of alpha-lactalbumin and other minor proteins in WPI.     

Enzymatic hydrolysis of heat-induced aggregates of whey protein isolate

The effects of heat-induced denaturation and subsequent aggregation of whey protein isolate (WPI) solutions on the rate of enzymatic hydrolysis was investigated. Both heated (60 °C, 15 min; 65 °C, 5 and 15 min; 70 °C, 5 and 15 min, 75 °C, 5 and 15 min; 80 °C, 10 min) and unheated WPI solutions (100 g L(-1) protein) were incubated with a commercial proteolytic enzyme preparation, Corolase PP, until they reached a target degree of hydrolysis (DH) of 5%. WPI solutions on heating were characterized by large aggregate formation, higher viscosity, and surface hydrophobicity and hydrolyzed more rapidly (P < 0.001) than the unheated. The whey proteins exhibited differences in their susceptibility to hydrolysis. Both viscosity and surface hydrophobicity along with insolubility declined as hydrolysis progressed. However, microstructural changes observed by light and confocal laser scanning microscopy (CLSM) provided insights to suggest that aggregate size and porosity may be complementary to denaturation in promoting faster enzymatic hydrolysis. This could be clearly observed in the course of aggregate disintegration, gel network breakdown, and improved solution clarification.     

Effect of stirring and seeding on whey protein fibril formation

The effect of stirring and seeding on the formation of fibrils in whey protein isolate (WPI) solutions was studied. More fibrils of a similar length are formed when WPI is stirred during heating at pH 2 and 80 degrees C compared to samples that were heated at rest. Addition of seeds did not show an additional effect compared to samples that were stirred. We propose a model for fibril formation, including an activation, nucleation, growth, and termination step. The activation and nucleation steps are the rate-determining steps. Fibril growth is relatively fast but terminates after prolonged heating. Two processes that possibly induce termination of fibril growth are hydrolysis of nonassembled monomers and inactivation of the growth ends of the fibrils. Stirring may break up immature fibrils, thus producing more active fibrils. Stirring also seems to accelerate the kinetics of fibril formation, resulting in an increase of the number of fibrils formed.     

Structural changes imposed on whey proteins by UV irradiation in a continuous UV light reactor

The objective of this study was to investigate the structural changes of whey proteins during exposure in a continuous-flow UV reactor. Varying UV irradiation dosages were obtained by controlling the flow rate and the mixing speed. Whey protein isolate (WPI) solutions at concentrations of 1% and 5% (w/v) were circulated at flow rates ranging from 30 to 800 mL·min(-1), and changes in physicochemical properties of the proteins were investigated. Intrinsic fluorescence spectra and surface hydrophobicity measurements suggested changes in the tertiary structure of the proteins with UV exposure. The UV treatment also increased the concentration of total and accessible thiol groups in 1% WPI solutions, while no change was measured in 5% WPI solutions. Size-exclusion chromatography demonstrated the formation of UV-induced aggregates and oxidation products (N-formylkynurenine and dityrosine) of aromatic amino acids. Furthermore, the UV-induced changes in protein conformation increased the susceptibility of whey proteins to pepsin hydrolysis.     

Influence of pH and ionic strength on heat-induced formation and rheological properties of soy protein gels in relation to denaturation and their protein compositions

The influence of pH and ionic strength on gel formation and gel properties of soy protein isolate (SPI) in relation to denaturation and protein aggregation/precipitation was studied. Denaturation proved to be a prerequisite for gel formation under all conditions of pH and ionic strength studied. Gels exhibited a low stiffness at pH >6 and a high stiffness at pH <6. This might be caused by variations in the association/dissociation behavior of the soy proteins on heating as a function of pH, as indicated by the different protein compositions of the dissolved protein after heating. At pH 3-5 all protein seems to participate in the network, whereas at pH >5 less protein and especially fewer acidic polypeptides take part in the network, coinciding with less stiff gels. At pH 7.6, extensive rearrangements in the network structure took place during prolonged heating, whereas at pH 3.8 rearrangements did not occur.     

Heat-induced gel formation by soy proteins at neutral pH

Heat-induced gel formation by soy protein isolate at pH 7 is discussed. Different heating and cooling rates, heating times, and heating temperatures were used to elucidate the various processes that occur and to study the relative role of covalent and noncovalent protein interactions therein. Gel formation was followed by dynamic rheological measurements. Heat denaturation was a prerequisite for gel formation. The gelation temperature (84 degrees C) was just above the onset denaturation temperature of glycinin. The stiffness of the gels, measured as the elastic modulus, G', increased with the proportion of denatured protein. An increase in G' was also observed during prolonged heating at 90 degrees C. This increase is explained by the occurrence of rearrangements in the network structure and probably also by further incorporation of protein in the network. The increase in G' upon cooling was thermoreversible indicating that disulfide bond formation and rearrangements do not occur upon cooling.     

Fractionation of whey protein isolate with supercritical carbon dioxide to produce enriched α-lactalbumin and β-lactoglobulin food ingredients

An environmentally friendly protein fractionation process using supercritical carbon dioxide (SCO(2)) as an acid was developed to produce enriched α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) fractions from whey protein isolate solutions containing from 2 to 10% WPI. This study investigated the effects of pH, temperature, WPI concentration, and residence time on the precipitation kinetics and recovery yields of individual whey proteins and the relative enrichment and composition of both protein fractions. At 5.5-34 MPa and 60-65 °C, solubilized SCO(2) decreased solution pH and induced the formation and precipitation of α-LA aggregates. Gel electrophoresis and HPLC of the enriched fractions demonstrated the production of ≥ 60% pure α-LA, and ≥ 70% pure β-LG, under various operating conditions, from WPI containing ～57% β-LG and 21% α-LA. The enriched fractions are ready-to-use food ingredients with neutral pH, untainted by acids and contaminants.